AJCS 6(6):995-1003 (2012)
ISSN:1835-2707
Comparative effects of two alkali stresses, Na2CO3 and NaHCO3 on cell ionic balance, osmotic
adjustment, pH, photosynthetic pigments and growth in oat (Avena sativa L.)
Zhan-Wu Gao1, Ji-Tao Zhang2, Zhuang Liu3, Qing-Tao Xu1, Xiu-Jun Li2 and Chun-Sheng Mu4*
1
Baicheng Normal University, Department of Geography, Baicheng 137000, China
Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130012, China
3
Baicheng City Academy of Agriculture Sciences, Ji Lin Province Research Institute of Sunflower, 137000, China
4
Institute of Grassland Science, Northeast Normal University, Key Laboratory of Vegetation Ecology of Ministry
of Education, Changchun 130024, China
2
*Corresponding author: [email protected]
Abstract
This study examines the comparative effects of NaHCO3 and Na2CO3 on young oat (Avena sativa L.) plants to elucidate the species’
physiological adaptive mechanisms to alkali stress. Factors considered are the intracellular influx and efflux of ions, ionic balance,
osmotic adjustment, pH homeostasis, photosynthetic pigments and growth. Results show that, Na2CO3 had stronger effects than
NaHCO3, and that with increasing concentrations of both stresses the plant showed rising Na+ influxes into the shoot resulting in Na+
ion toxicity. This is tolerated by Na+ sequestration in the vacuole; the accumulation mainly of Cl-, SO42- and the synthesis of high
concentrations of organic anions to maintain vacuolar ionic balance and, lastly by the synthesis of proline in the cytoplasm to avoid
dehydration. Moreover, Na2CO3 stress inhibits growth more strongly, compared to NaHCO3, because of the higher energy costs
associated with Na+ exclusion and compartmentalisation，the syntheses of organic anions, the syntheses of proline in the cytoplasm,
reduced photosynthetic capacity and increased membrane permeability. Compared to the shoot, although the root had a similar
response to both stresses, it showed a higher tolerance because high Na2CO3 stresses (>48 mmol L-1) resulted in significant increases
in root tissue pH, but did not affect the pH homeostasis of the shoot. Additionally, while both stresses decreased root dry weight, they
did not significantly affect root extension growth. This indicates that oat adopts an opportunistic guerrilla strategy by which it avoids
resource-poor patches of soil (e.g. high alkali) while preferentially exploiting more favorable habitats by maintaining root extension.
Keywords: alkali; ionic balance; Na2CO3 and NaHCO3 stresses; oat; shoot and root growth.
Abbreviations: ELR, electrolyte leakage rate; OA, organic acid.
Introduction
Alkali stress has been demonstrated in a number of reports
(Kawanabe and Zhu, 1991; El-Samad and Shaddad, 1996;
Campbell and Nishio, 2000; Hartung et al., 2002; Rao et al.,
2008; Wang et al., 2011; Ma et al., 2011). Previous studies
have suggested that alkali stress results mainly from levels of
the alkaline salts NaHCO3 and Na2CO3 (Shi and Yin, 1993; Shi
and Sheng, 2005; Yang et al., 2010; Liu et al., 2010). In some
areas, soil alkalisation is a severe problem. For example,
approximately 7% (6.7106 hm2) of the cultivated land in
northeast China has alkaline soils with only a few
alkali-tolerant halophytes being able to survive there
(Kawanabe and Zhu, 1991). Alkali stress usually involves a
combination of stresses, osmotic, ion-induced injury and high
pH (Munns, 2002; yang et al., 2008a; Chen et al., 2011). The
high-pH environment that surrounds the roots can greatly affect
the absorption of cations and inorganic anions and can also
disrupt the ionic balance and pH homeostasis of the tissues
(Yang et al., 2007, 2008b; Guo et al., 2010). The results are a
decrease in photosynthesis, damage to the membrane system
and finally a reduction in growth. Although the effects of
various mixed alkali stresses have been studied extensively in a
few plants (Shi and Yin, 1993; Hartung et al., 2002; Shi and
Wang, 2005; Yan et al., 2006; Guo et al., 2010; Radi et al.,
2012), these studies do not differentiate between the effects of
NaHCO3 and of Na2CO3 in isolation. Although these studies
provide a better understanding of the adaptive responses of
plants under mixed alkaline stress, analysis of the stresses
caused by the single alkali have not been adequately researched.
Examination of the effects of NaHCO3 alone, or of Na2CO3
alone, would seem to be a logical starting point for resolving
the demographic mechanisms underlying these complex
responses. Therefore, stress effects associated with NaHCO3 on
its own or with Na2CO3 on its own should be investigated as
thoroughly as those of mixed alkali stress, especially, in regard
to the responses of the major plant organs, the shoot and root.
The present study was conducted in an experimental area of
Northeast Normal University during the 2008 season. We
compare the separate effects of alkali stresses, NaHCO3 stress
and Na2CO3 stress on water potential, photosynthetic pigments,
ionic balance and tissue pH and growth in the shoot and root of
young oat seedlings, to enhance our understanding of the
mechanisms of alkali stress damage to plants and also those by
which they adapt to such alkali stress.
Results
Tissue pH and ELR analysis of shoot and root
Shoot tissue pH did not show significant differences between
NaHCO3 and Na2CO3 stresses but root tissue pH and
electrolyte leakage rate (ELR) were significantly higher under
Na2CO3 stress than under NaHCO3 stress (Table 1). The shoot
995
tissue pH under both stresses was closely similar to the controls
(Fig. 1A, B) suggesting that these stress intensities did not
affect the aboveground tissue pH. However, the root pH
showed a different behaviour. While NaHCO3 stress did not
affect root pH with increasing stress, middle and high Na2CO3
contents (≥72mmol L-1) did cause significant increases. Both
stresses increased the ELR but the extent of the increment
under Na2CO3 stress was much greater than under NaHCO3
stress (Fig. 1C). All the plants treated with 120 and 144 mmol
L-1 of Na2CO3 died so their pH values were not recorded.
increase in Cl- in the Na2CO3 was higher than in SO42- (Fig. 5A,
B, C, D). Both NO3 -and H2PO4- decreased with increasing
stress but the reductions with Na2CO3 were much greater than
with NaHCO3 (Fig. 5E, F, G, H). With increasing stress, both
low NaHCO3 (≤72mmolL-1) stresses and all the Na2CO3
stresses caused increases in organic acid. However, when the
NaHCO3 stresses were higher than 72 mmol L-1，OA showed
decreases (Fig. 5I, J).
Growth indices analysis of oat
In both shoot and root, proline concentrations were
significantly higher under Na2CO3 stress than under NaHCO3
stress while water contents were significantly lower (Table 1).
Both NaHCO3 and Na2CO3 stress decreased the water contents
of shoots and roots (Fig.6A, B) but the extent of the reductions
with increasing Na2CO3 stress were greater than with
increasing NaHCO3 stress. For shoot and roots, the changes in
water content were in opposite directions with proline being
increased by strong alkaline stress (>120mmolL-1in NaHCO3
and ≥96 mmolL-1 in Na2CO3) (Fig. 6C, D).
With increasing alkali stress, the shoot survival rate, number of
tillers per plant, root length, plant height, shoot dry weight and
root dry weight all decreased (Fig. 2A-F), but the extents of the
reductions were different. The effect on root length was
especially slight with the effect of NaHCO3 being not
significant and only the most concentrated Na2CO3 (144mmol
L-1) caused a significant decrease (Fig. 2C, table 1).
Furthermore, the reductions under Na2CO3 stress were much
greater than those under NaHCO3 stress (table 1).
Water and proline contents analysis of shoot and root
Discussion
Photosynthetic pigments analysis of leaves
Tissue pH
All photosynthetic pigments were significantly lower under
Na2CO3 than under NaHCO3 (Table 1). The effects of NaHCO3
stress on Chl a, Chl b and carotenoid were similar with all three
parameters decreasing but the extents of the reductions under
Na2CO3 stress were greater than those under NaHCO3. When
Na2CO3 stress was ≥ 120 mmolL-1 the plants died and the
contents of Chla , Chlb and Carotenoid were not recorded. The
same concentrations of NaHCO3 did not result in plant death
(Fig. 3).
Cations analysis of shoot and root
Each of the cations showed significant differences between the
NaHCO3 and Na2CO3 stresses (Table 1). In both the shoots and
the roots, the concentration ratios of Na+ and Na+ ⁄ K+ in the
Na2CO3 stressed plants compared to the NaHCO3 stressed
plants were significantly higher, while for K+ and Ca2+ the
concentration ratios were significantly lower (Table 1). Both
shoots and roots showed similar trends for these cations (Fig.
4A, B, C, D, G, H). With increasing stress, both the NaHCO3
and Na2CO3 stresses resulted in increased Na+ contents and a
decreased K+ contents, and finally in an increased Na+ ⁄ K+ ratio
(Fig. 4A, B, C, D, G, H). When NaHCO3 and Na2CO3 stresses
were compared, the extents not only of the increases in Na+ but
also of the reductions in K+ were much greater under Na2CO3
stress than under NaHCO3 stress (Fig. 4A, B, C, D, G, H). In
both shoot and root, although the Ca2+ content range was far
less than for either Na+ or K+, it showed a similar trend for
decrease compared to K+ with increasing alkali stress, also, the
extent of the Ca2+ decrease for Na2CO3 stress was greater than
for the NaHCO3 stress (Fig. 4E, F).
Anions analysis of shoot and root
All the anions showed significant differences between the
NaHCO3 and Na2CO3 stresses (Table 1). In both shoot and root,
compared to the NaHCO3 stress, the Na2CO3 stress showed
significantly higher concentrations of SO42- and organic acids,
while the Cl- , NO3 -and H2PO4- concentrations were
significantly lower (Table 1). In both shoot and root Cl- and
SO42- increased with increasing stress intensity for both
stressors but compared with the NaHCO3 stress, the extent of
Regardless of environmental pH, to maintain normal
metabolism it is important that plants can stabilise their tissue
pH (Yang et al., 2007). Our observations that shoot tissue pH
was similar to the control values under both alkali stresses and
that stress intensity increases did not have significant affects on
tissue pH suggests that oat is able to maintain a stable cell pH
(Fig.1A, B). However, it is surprising that when the stress level
rose above certain threshold values (48 mmol L-1 with Na2CO3;
72 mmol L-1 with NaHCO3), root pH was affected by increasing
levels of stress. This may be explained as that at above 48
mmol L-1 in Na2CO3 (or 72 mmol L-1in NaHCO3) the harmful
effects of high pH were opposed by pH adjustments outside the
roots (by extrusion of H+, OA, amino acids or CO2 produced by
root respiration) with the intracellular environment being
unaffected. However, when the stress intensity exceeded the
capacity for root adjustment (>48 mmol L-1 in Na2CO3 or >72
mmol L-1in NaHCO3,), the result was a reduction in
photosynthetic pigment content (Fig. 3), and a sharp increase in
ELR (Fig. 1C). This suggests that the stress may have
weakened the controls, leading to increases in pH. Meanwhile,
compared to the shoot, it was found that the effects of both
alkali stresses on root growth (i.e. root length and biomass) was
reduced, indicating that the root has a higher tolerance of
elevated pH. This deserves further investigation.
Growth indices
Our results show that the injurious effects of Na2CO3 on growth
were greater than those of NaHCO3 for the same alkali content
(Fig. 2), and this is consistent with previous reports (Shi and
Yin, 1993; Yang et al., 2007). The injurious effects of alkali are
commonly thought to be due to a combination of low water
potential, ion toxicity, and, in particular, to high-pH stress
(Munns, 2002). Firstly, the greater alkali stress due to Na2CO3
compared with NaHCO3 leads to severer reductions in
photosynthetic pigment content (Fig. 3) and a sharp increase in
ELR (Fig. 1C). These results indicate that high pH from
Na2CO3 stress may damage root cell structure and function
affecting such processes as the absorption of ions, damaging
photosynthetic pigments and membrane systems (e.g. increases
in ELR). This may be why growth under NaHCO 3 stress was
996
Table 1. Results of a two-way ANOVA of plant characteristics by stress category, and by stress gradient and their interaction.
Dependent variable
Stress category
Stress gradient
Category×Gradient
(A) ions and organic acid
shoot Na+ content
2053.806***
441.723***
194.087***
root Na+ content
1962.682***
448.914***
185.606***
shoot K+ content
594.499***
144.279***
32.183***
root K+ content
617.148***
151.240***
33.286***
shoot Ca2+ content
237.577***
75.229***
10.253***
root Ca2+ content
92.747***
135.584***
10.522***
shoot Na+ ⁄ K+ ratio
6103.667***
1186.216***
693.524***
root Na+ ⁄ K+ ratio
5843.242***
1178.227***
663.099***
shoot Cl- content
8730.910***
2111.533***
1501.335***
root Cl- content,
130.938***
186.921***
7.554***
shoot SO42- content
252.528***
155.270***
14.193***
root SO42- content
88.950***
73.563***
5.714**
shoot NO3– content
5849.245***
510.064***
121.803***
root NO3– content
5066.797***
1204.266***
206.504***
shoot H2PO4- content
216.028**
23.991***
10.599***
root H2PO4- content
174.224***
45.478***
7.879***
shoot organic acid content
1201.624***
73.181***
129.200***
root organic acid content
140.200***
26.039***
10.502***
(B) water and proline
shoot water content
29.525***
12.718***
2.522 n.s.
root water content
16.613***
4.594**
1.929 n.s.
shoot proline content
15.242**
62.169***
3.025*
root proline content
25.284***
142.933***
5.750**
(C) tissue pH, ELR , Chl, Car
shoot tissue pH
0.410 n.s.
0.105 n.s.
0.164 n.s.
root tissue pH
112.706***
22.139***
19.165***
electrolyte leakage rate (ELR)
72.665***
71.086***
4.010**
chlorophyll (Chl) a
84.845***
23.822***
8.851***
chlorophyll (Chl) b
86.057***
34.020***
8.783***
Carotenoid (Car)
142.607***
43.361***
21.962***
(D) growth index
survival rate
1768.559***
303.930***
215.895***
number of tillers per plant
24.523***
12.629***
3.669**
root length
3.679 n.s.
2.230 n.s.
0.315 n.s.
plant height
46.796***
30.127***
1.948 n.s.
shoot dry weight
26.597***
13.593***
3.485 n.s.
root dry weight
13.669**
13.946***
0.839 n.s.
Note: Numbers represent F values: * P ≤ 0.1; ** P ≤ 0.01; *** P ≤ 0.001; n.s., no significant.
less than under Na2CO3 stress. Next, to tolerate ion toxicity,
plants can exclude Na+ depending on a Na+ ⁄ H+ antiport,
synthesise organic anions to maintain ionic balance or
synthesise compatible solutes in the cytoplasm to prevent
dehydration. All of these cost energy and hence involve a
growth penalty. Thus, the higher Na+ toxicity under Na2CO3
stress than under NaHCO3 likely results in higher energy costs,
which may be another reason for the reduced growth under
NaHCO3 stress. Additionally, it is interesting that, compared to
the control, root length growth did not change significantly for
any NaHCO3 concentrations or for the low and medium
concentrations of Na2CO3 (<144mmolL-1). However, root dry
weight growth did decrease significantly under >96mmolL-1. It
is suggested that, under natural conditions, oat appears to use
an opportunistic guerrilla strategy whereby resources are
allocated in the direction of a more favourable microsite by
maintaining root extension rates even though root dry weight is
significantly decreased as a result of damage to the
photosynthetic pigments and the membrane systems (e.g.
increases of ELR). By this strategy, oat can avoid resource-poor
(e.g. high alkali patches of soil) while preferentially exploiting
more favorable habitats.
Ion toxicity and ion imbalance
Under high concentrations of either alkali, the first effect on the
plant is likely to be in the root due to high concentrations of
Na+ in the soil. The Na+ enters the roots passively via voltage
independent nonselective cation channels and possibly via
other Na+ transporters such as some members of the
high-affinity K+ transporter (HKT) family (Munns and Tester,
2008), resulting in Na+ ion toxicity in shoot. The similar radii
of the hydrated ions of Na+ and K+ makes them difficult to
discriminate, and this is the basis of Na+ toxicity (Blumwald,
2000). Under both alkali stresses, Na+ competes with K+ for
uptake into the roots (Munns, 2002; Munns and Tester, 2008).
Compared to the same stress concentrations of NaHCO3 and
Na2CO3, the latter has the higher concentration of Na+ resulting
in increased Na+ accumulation (associated with decreased K+
accumulation) (Fig. 4ABCD). Also, the increased Na+ in the
stressed plant may induce a decrease in Na+ exclusion, which
normally enables the plant to avoid or defer ion toxicity
problems. Many plant species have a Na+ exclusion mechanism
that depends on Na+ ⁄ H+ antiport, such as salt overly sensitive 1
997
NaHCO3
[B]
Na2CO3
Shoot tissue pH
c
6.0
a
a
a
a
a
a
a
a
8.0
ab
a
a
b
b
c
b
c
c
a
a
6.0
4.0
4.0
2.0
2.0
0.0
Root tissue pH
[A]
8.0
0.0
0
48
72
96
120
144
0
48
72
[C]
96
120
144
e
80
ELR (%)
d
c
60
b
40
e
de
b
cd
bc
ab
20
a
a
0
0
48
72
96
120
144
Alkalinity (mM)
Fig 1. Effects of NaHCO3 and Na2CO3 stress on (A) shoot tissue pH, (B) root tissue pH, (C) ELR (electrolyte leakage rate) in oat
shoots. The 4-week-old oat seedlings were treated with NaHCO3 stress (pH 8.03-8.26) and Na2CO3 stress (pH 9.91-11.27) for 9 days.
In each column, the data markers identified with the same letters are not significantly different (P < 0.05) according to a Duncan test.
The error bars represent ± standard error (n = 3) of three replicates. Shoot represents aboveground part of plant.
Fig 2. Effects of NaHCO3 and Na2CO3 stress on (A) survival rate, (B) number of tillers per plant, (C) root length, (D) plant height, (E)
shoot dry weight, (F) root dry weight in oat. The 4-week-old oat seedlings were treated with NaHCO3 stress (pH 8.03-8.26) and
Na2CO3 stress (pH 9.91-11.27) for 9 days. In each column, the data markers identified with the same letters are not significantly
different (P < 0.05) according to a Duncan test. The error bars represent ± standard error (n = 3) of three replicates. DW, dry weight;
Shoot represents aboveground part of plant.
998
(SOS1), which exchanges cytoplasmic Na+ with external H+
(Zhu, 2003; Munns and Tester, 2008). The exchange activity
relies on the transmembrane proton gradient achieved by
H+-ATPase (Zhu, 2003). Compared to NaHCO3, the same
concentration of Na2CO3 has a higher pH, and its relatively lack
of external protons may weaken the exchange activity of the
Na+ ⁄ H+ antiport on the root plasma membrane (Munns and
Tester, 2008), possibly reducing the exclusion of Na+ from the
rhizosphere and enhancing plant accumulation of Na+ (Fig. 4).
In addition, compared to Na+ and K+, other ions, especially
Ca2+ can also be influenced by both alkali stresses each of
which exhibits decreasing trends with increasing concentrations.
However, the extent of this effect is significantly higher in
Na2CO3 than in NaHCO3. This is firstly because, higher Na+ in
the shoot is influenced by Na2CO3 replacement of more
intra-cellular Ca2+ which results in substantial Ca2+ loss (Fig.
4EF ); secondly, the high-pH environment of the roots with
Na2CO3 can cause some ions, such as Ca2+ to precipitate
directly (Shi and Zhao, 1997); thirdly, Ca2+ can be influenced in
the plant by higher contents of organic acid (mainly oxalic acid)
(Fig. 5IJ) through the formation of calcium oxalate (an
insoluble crystal) from oxalic acid and Ca2+. In the root, the
high concentration of Na+ causes this ion to move in the
symplast across the endodermis where it is released from the
stelar cells to the stelar apoplast, from where it moves through
the xylem in the transpiration stream, finally reaching the shoot
where the Na+ toxicity occurs (Munns and Tester, 2008). The
main site of Na+ toxicity for most plants is in the leaf blade.
Here, it can be tolerated by anatomical adaptations and by
intracellular partitioning with the Na+ remaining in the cell
being sequestered in the vacuoles to avoid Na+ toxicity in the
cytosol (Serrano and Rodriguez-Navarro, 2001; Munns, 2002;
Zhu, 2003).
Na+ toxicity in the cytosol (Serrano and Rodriguez-Navarro,
2001; Zhu, 2003) and accumulate inorganic anions and
synthesise organic anions to maintain vacuolar ionic balance
(Yang et al., 2007), they may also synthesise compatible low
molecular weight organic solutes (compatible solutes) in the
cytoplasm to maintain osmotic equilibrium and so prevent
dehydration and protect biomacromolecules (Parida and Das,
2005). The amino acid proline is one of the most widely
distributed compatible solutes occurring in many organisms
from bacteria to higher plants (Flowers et al., 1977). In many
halophytes, proline occurs at sufficiently high concentrations in
their leaves (over 40 mM on a tissue water basis) to contribute
significantly (over 0.1 MPa) to cell osmotic potential (Fricke,
2004). Thus, in our study it is clear that Na+ concentration
increases in the vacuoles (which also accumulate inorganic and
organic anions to maintain ionic balance) and as alkali stress
increases, proline concentration increases to prevent
cytoplasmic dehydration (Fig. 6). Furthermore, for the same
intensity of the two alkali stresses the especially high
concentrations of Na+ with Na2CO3 lead to relatively high
concentrations of proline (Fig. 6). In particular, the induction of
proline synthesis is also related to the high pH value associated
with alkalinity. The involvement of proline accumulation in the
damage caused by alkali stress is another aspect that requires
further investigation. Apart from the above-mentioned osmotic
adjustment which comes with an energy cost, a quick and
energetically economical way of reducing cell water potential
to avoid osmotic stress is to allow decreases in water content
(Fig. 6AB). In summary, the key features pertaining to osmotic
adjustment are an ability to accumulate Na+ and organic acids
in the vacuoles and to accumulate large amounts of proline in
the cytoplasm.
Materials and methods
Ion balance
Plant materials
When compartmentalisation of Na+ in the vacuoles takes place,
plants usually accumulate inorganic anions, such as Cl-, and
SO42-, or they synthesise organic anions to maintain the ionic
balance (Yang et al., 2007). This fits with our study (Fig. 5)
where the concentrations of inorganic anions under NaHCO3
stress were significantly lower than those under Na2CO3 stress
of the same intensity. This suggests that the relatively high pH
caused by Na2CO3 stress may inhibit the uptake of anions such
as NO3- and H2PO4- (Fig. 5EFGH). Meanwhile, Cl- and SO42- in
relatively high concentrations in both alkalis make higher
contributions to the ion balance. Additionally, we find that,
with stress concentration increases in the plant, OA is strongly
increased only with Na2CO3 whereas with NaHCO3 it is
decreased. This suggests that under Na2CO3 stress, OA is the
dominant ion in the maintenance of equilibrium. This may be
an alkali resistance mechanism involving two components,
intracellular pH adjustment and extracellular pH adjustment
(also possibly, pH adjustment in the root-external
microenvironment). The OA synthesised might also be
transported to roots for pH regulation. The process of pH
adjustment may occur outside the root or in the root apoplast,
or both. Therefore, the type of cells involved in pH adjustment
may be epidermal, cortical or xylem parenchyma cells. The
mechanism of pH adjustment may involve the exudation of a
buffer compound, such as H+, OA, amino acids or even CO2
produced by root respiration, or other factors not considered
above.
Osmotic adjustment
When plants compartmentalise Na+ into the vacuoles to avoid
Oat (Avena sativa L.) is an annual, graminaceous plant which is
very tolerant of drought, cold, saline and alkali stress and
mineral deficiency. The oat cultivar No.2 baiyanmai (numbered
species is B07046-2-4-1-6 in Baicheng City Academy of
Agriculture Sciences, China) was used. This cultivar is
characterised by early maturation, high salt and alkali tolerance
and high disease resistance (Wei et al., 2007). It has a high
grain yield which rises to over 2300 kg/hm2. Its protein and fat
contents are 16.6% and 5.6%, respectively (Wei et al., 2007).
The plant can mature in live culms and provides both grain and
straw.
Cultivation
Seeds were sown in 20-cm diameter plastic pots containing 3
kg of washed sand. The pots were watered daily with sufficient
Hoagland nutrient solution. Each pot contained 25 seedlings.
All pots were placed outdoors and protected from the rain. The
experiment was carried out in an experimental area of the
Northeast Normal University during the 2008 growth season.
Design of the simulated alkaline conditions
Two alkaline salts, NaHCO3 and Na2CO3 were applied
separately to create two stress groups. Within each group, six
concentrations were used: 0, 48, 72, 96, 120 and 144 mmol L-1.
Treatments in the NaHCO3 stress group were labeled A1–A6
and those in the Na2CO3 stress group were labeled B1–B6. A1
and B1 were the controls. There were three replicates per
treatment.
999
Fig 3. Effects of NaHCO3 and Na2CO3 stress on the (A) chlorophyll (Chl) a, (B) chlorophyll (Chl) b in oat. The 4-week-old oat
seedlings were treated with NaHCO3 stress (pH 8.03-8.26) and Na2CO3 stress (pH 9.91-11.27) for 9 days. In each column, the data
markers identified with the same letters are not significantly different (P < 0.05) according to a Duncan test. The error bars represent
± standard error (n = 3) of three replicates. FW, fresh weight; Shoot represent aboveground part of plant.
Stress treatments
Measurement of chlorophyll
The stress treatments were applied when the seedlings were
four weeks old. Thirty-six pots of uniform seedlings were
divided randomly into 12 sets of 3 pots. Two sets were used for
the controls and the remaining 10 sets were used for the stress
treatments giving three replicate pots per treatment. The treated
pots were watered daily between 16.00–18.00 h with excess of
a nutrient solution that contained the appropriate stress salts.
The control plants were watered with nutrient solution at the
same time. The duration of stress treatment was nine days.
After 9 days of stress treatment, fresh healthy leaves were cut
into small segments to determine the concentrations of
chlorophylls a and carotenoid b according to Arnon (1949).
Physiological indices measurements
Measurement of tissue pH
To determine tissue pH, fresh shoots and roots were washed
thoroughly three times with neutral deionised water, followed
by surface-drying with filter paper. They were then crushed and
the pH of the expressed sap measured with a digital pH meter
PHS-3C; Shanghai precision & scientific instruments, Shanghai,
China.
Measurement of the electrical conductivity of the leaves
Membrane permeability is reflected in a ‘relative electrical
conductivity’, which is defined as the ratio of the electrical
conductivity of leaves with intact membranes to those with
membranes destroyed by a boiling water treatment. Electrolyte
leakage rate (ELR) was determined as described by Lutts et al.,
(1996).
Measurement of growth indices
All plants were harvested in the morning after the final
treatment. The number of tillers per plant was recorded. The
plants were first washed with tap water and then with distilled
water. For each plant, the roots and shoots were separated and
their fresh weights (FW) and the lengths of their shoots and the
total root length per plant were determined. The samples were
then oven-dried at 105 ℃ for 15 min before being
vacuum-dried at 80℃ to constant weight. The shoot and root
dry weights (DW) were recorded. The water content (WC) of
both parts was calculated using the formula WC= (FW-DW) /
FW. Survival rate (SR) was expressed using the formula: SR= n
/ N where n is the number of plants surviving from the total of
N plants.
Measurement of ions
Dry samples (0.1 g) of shoot and root were treated with 20 mL
of deionised water at 100℃ for 1 h and the resultant extract
was used to determine the contents of inorganic ions and
organic acids (OA).The contents of NO3-, Cl- , SO42-, H2PO4were determined by ion chromatography using a DX-300 ion
chromatographic system with an AS4A-SC ion-exchange
column and a CDM-II electrical conductivity detector (mobile
1000
Fig 4. Effects of NaHCO3 and Na2CO3 stress on (A) shoot Na+
content, (B) root Na+ content, (C) shoot K+ content, (D) root K+
content, (E) shoot Ca2+ content, (F) root Ca2+ content, (G)
shoot Na+ ⁄ K+ ratio, (H) root Na+ ⁄ K+ ratio in oat. The
4-week-old oat seedlings were treated with NaHCO3 stress (pH
8.03-8.26) and Na2CO3 stress (pH 9.91-11.27) for 9 days. In
each column, the data markers identified with the same letters
are not significantly different (P < 0.05) according to a Duncan
test. The error bars represent ± standard error (n = 3) of three
replicates. DW, dry weight; Shoot represent aboveground part
of plant.
phase: Na2CO3 ⁄ NaHCO3 = 1.7 ⁄ 1.8 mmol L-1; DIONEX,
Sunnyvale, CA, USA). Na2CO3 ⁄ NaHCO3=1.7 / 1.8 mmol L-1.
The levels of the organic acids were also determined by ion
chromatography using an DX-300 ion chromatographic system
with an ICE-AS6 ion-exclusion column, CDM-II electrical
conductivity detector and an AMMS-ICE II MicroMembrane
suppressor (mobile phase: 0.4 mmol L-1 heptafluorobutyric acid;
DIONEX). An atomic absorption spectrophotometer (TAS-990;
Purkinje General, Beijing, China) was used to determine the
levels of Na+, K+ and Ca2+.
Measurement of proline
The dried samples were homogenised to determine free proline
contents which was assayed using the acid-ninhydrin method
(Zhu et al. 1983).
Fig 5. Effects of NaHCO3 and Na2CO3 stress on (A) shoot Clcontent, (B) root Cl- content, (C) shoot SO42- content, (D) root
SO42- content, (E) shoot NO3– content, (F) root NO3– content,
(G) shoot H2PO4- content, (H) root H2PO4- content, (I)shoot
OA (organic acid) content, (J) root OA (organic acid) content
in oat. The 4-week-old oat seedlings were treated with
NaHCO3 stress (pH 8.03-8.26) and Na2CO3 stress (pH
9.91-11.27) for 9 days. In each column, the data markers
identified with the same letters are not significantly different (P
< 0.05) according to a Duncan test. The error bars represent ±
standard error (n = 3) of three replicates. DW, dry weight;
Shoot represent aboveground part of plant.
1001
Fig 6. Effects of NaHCO3 and Na2CO3 stress on the (A) shoot water content, (B) root water content, (C) shoot proline content, (D)
root proline content in oat. The 4-week-old oat seedlings were treated with NaHCO3 stress (pH 8.03-8.26) and Na2CO3 stress (pH
9.91-11.27) for 9 days. In each column, the data markers identified with the same letters are not significantly different (P < 0.05)
according to a Duncan test. The error bars represent ± standard error (n = 3) of three replicates. Shoot represent aboveground part of
plant.
Statistical analyses
Statistical analysis of the data was performed using the
statistical program SPSS 13.0 (SPSS, Chicago, USA). All data
are represented by an average of three replicates and their
standard errors (SE). Data were analysed by one-way and
two-way ANOVA. The treatment mean values for the same
organ under either alkali stress were compared by post-hoc
Duncan tests. The term significant indicates differences at P <
0.05.
Acknowledgments
Financial support was provided by a collaborative grant from
the National Science Foundation (30471231), the Project
Foundation from the Education Ministry of China (106063), the
Specialized Research Fund for the Doctoral Program of Higher
Education (20050200006), and the National TCM Main Project
in 11th Five-Year-Period (2008BADB3B09) and the Science
and Technology Project of Jilin Province (20080563).
References
Arnon DI (1949) Copper enzymes in isolated chloroplasts.
Polyphenooxidase in Beta vulgaris. Plant Physiol. 24:1–15
Blumwald E (2000) Sodium transport and salt tolerance in
plants. Curr Opin Cell Biol. 12: 431–434
Campbell SA, Nishio JN (2000) Iron deficiency studies of
sugar beet using an improved sodium bicarbonate-buffered
hydroponics growth system. J Plant Nutr. 23: 741–757
Chen W, Feng C, Guo W, Shi D, and Yang C (2011)
Comparative effects of osmotic-, salt- and alkali stress on
growth, photosynthesis, and osmotic adjustment of cotton
plants. Photosynthetica. 49: 417-425
El-Samad HMA, Shaddad MAK (1996) Comparative effect of
sodium carbonate, sodium sulphate, and sodium chloride on
the growth and related metabolic activities of pea plants. J
Plant Nutr. 19: 717–728
Flowers TJ, Troke PF, and Yeo AR (1977) The mechanism of
salt tolerance in halophytes. Annu Rev Plant Physiol. 28:
89–121
Fricke W (2004) Rapid and tissue-specific accumulation of
solutes in the growth zone of barley leaves in response to
salinity. Planta. 219: 515–25.
Guo LQ, Shi DC and Wang DL (2010) The key physiological
response to alkali stress by the alkali-resistant halophyte
puccinellia tenuiflora is the accumulation of large quantities
of organic acids and into the rhyzosphere. J Agron Crop Sci.
196:123–135
Hartung W, Leport L, Ratcliffe RG, Sauter A, Duda R, and
Turner NC (2002) Abscisic acid concentration, root pH and
anatomy do not explain growth differences of chickpea
(Cicer arietinum L.) and lupin (Lupinus angustifolius L.) on
acid and alkaline soils. Plant Soil. 240: 191–199
Kawanabe S, and Zhu TC (1991) Degeneration and
conservation of Aneurolepidium chinense grassland in
Northern China. J Jpn Grassl Sci. 37: 91–99
Liu J, Guo WQ, and Shi DC (2010) Seed germination, seedling
survival, and physiological response of sunflowers under
saline and alkaline conditions. Photosynthetica. 48: 278-286
Lutts S, Kiner JM, and Bouharmont J (1996) NaCl-induced
senescence in leaves of rice (Oryza sativa L.) cultivars
differing in salinity resistance. Ann Bot. 78: 389–398
Ma Y, Guo LQ, Wang HX, Bai B, and Shi DC (2011)
Accumulation, distribution, and physiological contribution of
oxalic acid and other solutes in an alkali-resistant forage
plant, Kochia sieversiana, during adaptation to saline and
alkaline conditions. J Plant Nutr Soil Sc. 174: 655–663
1002
Munns R (2002) Comparative physiology of salt and water
stress. Plant Cell Environ. 25: 239–250
Munns R, and Tester M (2008) Mechanisms of salinity
tolerance. Annu Rev Plant. Biol. 59: 651–681
Parida AK, and Das AB (2005) Salt tolerance and salinity
effects on plants: a review. Ecotoxicol Environ Saf. 60:
324-349
Radi AA, Abdel-Wahab DA, Hamada AM (2012) Evaluation
of some bean lines tolerance to alkaline soil. J Biol Earth Sci.
2: B18-B27
Rao PS, Mishar B, Gupta SR, Rathore A (2008) Reproductive
stage tolerance to salinity and alkalinity stresses in rice
genotypes. Plant Breed. 127: 256–261
Serrano R, and Rodriguez-Navarro, A (2001) Ion homeostasis
during salt stress in plants. Curr Opin Cell Biol. 13: 399–404
Shi DC, and Sheng YM (2005) Effect of various salt–alkaline
mixed stress conditions on sunflower seedlings and analysis
of their stress factors. Environ Exp Bot. 54: 8–21
Shi DC, and Wang DL (2005) Effects of various salt–alkali
mixed stresses on Aneurolepidium chinense (Trin.) Kitag.
Plant Soil. 271: 15–26
Shi DC, and Yin LJ (1993) Difference between salt (NaCl) and
alkaline (Na2CO3) stresses on Puccinellia tenuiflora (Griseb.)
Scribn et Merr. plants. Acta Bot Sin. 3: 144–149
Shi DC, and Zhao KF (1997) Effects of NaCl and Na2CO3 on
growth of Puccinellia tenuiflora and on present state of
mineral elements in nutrient solution. Acta pratacu sin. 6:
51–61 (in Chinese)
Wang XP, Geng SJ, Ri YJ, Cao DH, Liu J, Shi DC, Yang CW
(2011) Physiological responses and adaptive strategies of
tomato plants to salt and alkali stresses. Sci Hortic. 130:
248–255
Wei LM, Guo LC, Sha L, Deng LG (2007) Breeding report on
new variety of oat Baiyan 2. Crop Research. 3: 341-342
Yan H, Zhao W, Yin SJ, Shi DC, and Zhou DW (2006)
Different physiological responses of Aneurolepidum
chinense to NaCl and Na2CO3. Acta Pratacult Sin. 15: 19–55
(in Chinese with an English abstract)
Yang C, Li C, Zhang M, Liu J, Ju M, and Shi D (2008a) Ion
and pH homeostasis in the shoot of wheatgrass (Triticum
aestivum-Agropyron intermedium) under salt and alkali
stresses. Chin J Appl Ecol. 19: 1000–1005 (in chinese)
Yang C, Shi D, and Wang D (2008b) Comparative effects of
salt stress and alkali stress on growth, osmotic adjustment
and ionic balance of an alkali-resistant halophyte Suaeda
glauca (Bge.). Plant Growth Regul. 56: 179–190
Yang CW, Chong JN, Kim CM, Li CY, Shi DC, and Wang DL
(2007) Osmotic adjustment and ion balance traits of an alkali
resistant halophyte Kochia sieversiana during adaptation to
salt and alkali conditions. Plant Soil. 294: 263–276
Yang CW, Guo WQ, and Shi DC (2010) Physiological roles of
organic acids in alkali-tolerance of the alkali-tolerant
halophyte chloris virgata. Agron J. 102: 1081–1089
Zhu JK (2003) Regulation of ion homeostasis under salt stress.
Curr Opin Plant Biol. 6: 441–445.
1003